Biology Reference
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11. Calculate the volume of the Cy3 solution (20 mmol/L, i.e.,
20 nmol/
L) containing the amount of Cy3 calculated in step
10, using the formula:
μ
volume of 20 mmol/L Cy3 solution( L)
μ=
amount of
Cy3 (nmol) / 20nmol/ L
μ
L)
12. Add the calculated volume of 20 mM Cy3 to the reaction
mixture.
13. Mix vigorously by pipetting (see Note 17).
14. Spin down the sample in a microcentrifuge.
15. Repeat steps 13 and 14 to make sure the solution is really
homogeneous and is located completely at the bottom of the
microcentrifuge tube.
16. Incubate at 37°C for 30 min in the dark.
17. Calculate the required volume of sample buffer I to adjust the
total volume of the labeling reaction mixture to 445.5
(e.g., 400 nmol Cy3/20 nmol/
μ
L = 20
μ
μ
L,
using the formula:
volume of sample buffer I( L)
μ =
445.5 L 250 L
10 L volume of Cy3
calculated in step 11
μ−
μ−
μ−
18. Stop the reaction by adding the volume of sample buffer I
calculated in step 17 to the reaction mixture.
19. Mix vigorously by pipetting.
20. Add 4.5
L of Pharmalytes (pI range corresponding to pI
range of IEF strips used). Total volume is now 450
μ
μ
L, corre-
sponding to the maximal volume applicable.
21. Add 9.0 mg of solid DTT for a fi nal DTT concentration of
65 mM DTT.
22. Mix vigorously by pipetting.
23. Spin down the sample in a microcentrifuge.
24. The sample with a total volume of 450
L is now ready for
isoelectric focusing. The sample can be stored frozen in the
dark for up to 1 month, preferably at −70°C.
μ
3.8. Preparative 2D
Gel Separation
It is essential to use the same type of electrophoretic devices as
used for the analytical gels. Otherwise, an unambiguous assign-
ment of corresponding spots between the analytical and prepara-
tive gels may be impossible. For isoelectric focusing of preparative
IEF strips, use the parameters given in Table
3
.
Appropriate precautions are strongly recommended in all steps
to avoid contamination with human keratin (see Note 18).
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